Neurosurgeons have long sought methods to illuminate brain cancer cells to identify cancer foci and distinguish cancer from normal tissue in real time during tumor resection operations. A bioconjugate composed of chlorotoxin (CTX), a peptide discovered from the Leiurus quinquestriatus scorpion, and near infrared fluorescent (NIRF) molecules, such as Cy5.5, (“tumor paint”) clearly identifies tumor foci with high sensitivity (M. Veiseh, et al., “Tumor Paint: A Chlorotoxin:Cy5.5 Bioconjugate for Intra-Operative Visualization of Cancer Foci,” Cancer Research 67(14):6882-88, 2007). CTX was originally selected for these studies because it preferentially binds to glioma cells compared with normal brain tissue (L. Soroceanu, et al., “Use of Chlorotoxin for Targeting of Primary Brain Tumors,” Cancer Research 58:4871-4879, 1998). Because the CTX target appears to be shared by multiple other cancer types, CTX:Cy5.5 effectively illuminated prostate, colon, sarcoma, medulloblastoma, and other types of solid tumors (M. Veiseh 2007).
CTX is a 36 amino acid peptide with four disulfide bridges that confer a high degree of three dimensional structure to the polypeptide. CTX has three lysine residues at positions 15, 23, and 27 that have been utilized for conjugation to NHS-ester modified Cy5.5 and other fluorescent molecules. The resultant bioconjugate is a mixture of typically 75-85% mono-labeled peptide at position 27 and lesser amounts of di- and tri-labeled peptide conjugated to Lys 15 and Lys 23. Although it is possible to have mixtures approved by the Food and Drug Administration (FDA) and similar regulatory agencies elsewhere, commercialization is potentially hindered as it is expensive and difficult to match the ratio of mono-, di- and tri-labeled batches in the future.
A need exists for a polypeptide having chlorotoxin's advantageous properties and having a single lysine residue for conjugation with diagnostic or therapeutic agents to provide a single, homogenous new molecular entity. The present invention seeks to fulfill this need and provides further related advantages.